Bioactive water fraction from Gomphostemma niveum

ABSTRACT

The present invention relates to a novel bioactive water fraction obtained from the leaves of an herb named  Gomphostema niveum  commonly found in North Eastern part of India, and which is useful for inhibiting the growth of malarial parasite  Plasmodium falciparum . The present invention also relates to a method for the extraction of said bioactive fraction. The present invention also provides methods for treatment of malaria using such bioactive water fractions and use of such bioactive water fractions for the treatment of malaria.

FIELD OF INVENTION

The present invention relates to a novel bioactive water fractionobtained from the leaves of an herb name Gomphostemma Niveum commonlyfound in North Eastern part of India, and which is useful for inhibitingthe growth of malarial parasite Plasmodium falciparum. The presentinvention also relates to a method for the extraction of said bioactivefraction. The present invention also provides methods for treatment ofmalaria using such bioactive water fractions and use of such bioactivewater fractions for the treatment of malaria.

BACKGROUND OF THE INVENTION AND PRIOR ART

Malaria is the most prevalent among the insect borne disease accountingfor more than two million deaths out of 400 million cases reported eachyear (Greenwood et. al., Nature 9415), 670(2002)) caused by Plasmodiumfalciparum. (Depote et al. Trends in parasitology (20), 4, 165-198,(2004)). More than half of the world's population lives in areas wherethey remain at risk of malarial infection (Sachs et at Nature, (415),686 (2002). The derivation of important anti-malarial compound startedwith the discovery of cinchona bark powder (quinine) (Brooking, G B106430, (1917)) in 1820. Subsequently post World War-I was a period ofintense work in maintaining such ethnobotanical records in which the useof quinine has remained the drug of choice of malaria. In 1940 anotheranti-malarial drug chloroquine (Ringwald et al, Bulletin of the worldhealth organization, 77(1), 34, (1999)) was synthesized and untilrecently this was the only drug used for the treatment of malaria.Unfortunately, after an early success, the malarial parasite especiallyP. falciparum became resistant to chloroquine. Treatment of chloroquineresistant malaria (White et al. Delaying anti-malarial drug resistancewith combination chemotherapy Parasitologia, (41), 301-308, (1999)) wasdone with alternative drug or drug combination (Tulp et. al, Drugdiscovery today (9)450, (2004)) which are rather expensive and sometimestoxic. Furthermore, these combinations are not always dependent uponpharmacokinetic principle due to inadequate knowledge of metabolism andmechanism of action of most anti-malarial drugs. In the last few years aplant based anti-malarial drug isolated from the Chinese plantArtimisinin annua (Klayman, Science (228), 1049, (1985)), Bharel et al.Fitoterapia, (67), 387-399, (1996)) proved to be very active against P.falciparum. However, the use of Artemisinin, an endoperoxidesesquiterpene lactone is somewhat limited because of its relatively highcost, limited production of GMP standards and reports of toxicity(Haynes et al. Curr. Opin. Infect. Dis, (14), 719-726, (2001)). Thecurrent route for the total synthesis of Artemisinin (Qinghaosu) (U.S.Pat. No. 6,710,074 (2004) is too complex for commercial production(Schmid et al J. Am. Chem. Soc. (105), 624-625, (1983). It is currentlyprepared by large scale extraction from Artemisia annua (sweet warwood)and derivative such as artemether, artesunate, arteether anddihydroartemisinin are prepared semi synthetically from the purifiedextract. Moreover, difficulties have also been encountered in theproduction of high quality material (Haynes et al. Curr. Opin. Infect.Dis., (14), 719-726, (2001)).

Thus, the widespread resistance of malarial parasite to the majority ofdrugs, necessitates the development of new drug (Wiesner et al. Angew.Chem. Int: Ed. (42), 5274-5293, (2003)) which should be structurallydifferent from classical compounds with novel mechanism of action toovercome the resistance problem. Recently Ihara et. al disclosed aboutcompounds having anti-malarial activity (U.S. Pat. No. 6,710,074 (2002))from synthesis. In recent past different classes of compound are alsoreported having anti-malarial activity like substituted 1,2,4 trioxane(U.S. Pat. No. 6,737,438 (2002)), flavonoids (WO2004000306 (2003)),naphthylisoquinoline (U.S. Pat. No. 6,627,641 (2003)), indoloquinazoles(U.S. Pat. No. 6,531,487 (2003)), trioxolanes (U.S. Pat. No. 6,486,199(2002)), betacarboline alkaloids (U.S. Pat. No. 6,143,756 (2000)),vocamine (WO 9948501(1999)), acetyl glucosamine derivatives.(DE3220426(1983)) and so on. Recently many patents like U.S. Pat. No.6,710,074, WO2004000319, U.S. Pat. No. 5,264,726, US2003212098,WO2004000306, EP1076057, WO9948501, U.S. Pat. No. 5,290,553, U.S. Pat.No. 6,143,756 and U.S. Pat. No. 6,627,641 disclosed compounds havinganti Plasmodium falciparum activity from natural origin mainly plants.Hence the ethnopharmacologial approach for the search of newanti-malarial has proved to be more predictive.

As a part of ongoing research programmes on bioresources, we havescreened large number of plants from the north eastern region for theanti-malarial inhibition studies. Plant selection was mainly based onvarious criteria such as chemotaxonomic data, field observation andrandom collection.

Malaria is caused by protozoa of the genus Plasmodium. Because of itsprevalence, virulence and drug resistance, it is the most serious andwidespread parasitic disease encountered by mankind. The inadequatearmory of drugs in widespread use for the treatment of malaria and lackof affordable new drugs are the limiting factors in the fight againstmalaria, which underscores the continuing need of research fordevelopment of new drugs.

There is therefore a need to develop new active compounds as analternative to chloroquine and especially from artemisinin, a plantbased anti-malarial drug isolated from the Chinese plant Artemisiaannua. The urgent need is to investigate the natural source with newethanophramacological approach for the search of new anti-malarialespecially from the North Eastern part of India, which has not beenscreened so far. Another need is to develop suitable method for theextraction, isolation and identification of bioactive principles formthe plant extract having the anti-malarial properties. The recentlydeveloped new isolation and characterization techniques together withdevelopment of new pharmacological testing have led to interest inplants as a source of new drugs. However, a promising approach is neededto use these agents as templates for designing new derivatives withimprove properties.

OBJECTS OF THE PRESENT INVENTION

The main objective of the invention is to develop a novel anti-malarialagent inhibiting the growth of chloroquine resistant and chloroquinesensitive malarial parasite from the natural source.

Another objective of the invention is to investigate the anti-malarialproperties of Indian medicinal plant from the North Eastern region ofIndia for the treatment of malaria (selection of the plant is based onchemotaxonomic data).

More particular objective is to develop a simple and efficient methodfor the extraction, isolation and identification of the activecomponents from the Indian medicinal plants.

Yet another objective is to perform in vitro bioassay of crude extractsagainst the growth of malarial parasite.

Further objective of the invention is to develop a reproduciblefingerprinting HPLC method for the routine monitoring of the crudeextract.

Still another objective of the invention is to provide a suitabletechnique for the identification, evaluation and monitoring of in vitroanti-malarial activity.

Another objective of the invention is to perform the temperaturestability studies for the different crude extract, this will be helpfulfor the storage of bioactive fraction.

Yet another objective is to evaluate the various routes ofadministration and to establish their stability in body fluids, toxicityand capacity to reach infected tissue in an adequate concentrationduring in vivo analysis.

STATEMENT OF INVENTION

The present invention therefore provides a novel bioactive waterfraction obtained from the leaves of Gomphostemma Niveum.

The present invention also provides a method for the preparation of anovel bioactive fraction from Gomphostemma Niveum comprising air dryingleaves of the plant, powdering the air dried leaves and subjecting thepowder to extraction with water filtering the extract and concentratingto dryness under reduced fraction to obtain the bioactive water extract.

In one embodiment of the invention, water extraction is carried outthree times.

In another embodiment of the invention, extraction is carried out bySoxhlet extraction method at reflux temperature for eight hours.

In yet another embodiment of the invention, extraction is carried outusing triple distilled water.

In yet another embodiment of the invention, the filtered extract isconcentrated to dryness under reduced pressure of about 86 psi at atemperature of about 46° C. using a rotary evaporator.

In another embodiment of the invention, the water extract obtained isfurther fractionated by liquid-liquid extraction using solvents selectedfrom the group consisting of hexane, diethyl ether, chloroform andethanol on the basis of increasing polarity; the extracts then beingconcentrated to dryness under reduced pressure of about 150 psi and at atemperature of about 30° C.

The present invention also provides a pharmaceutical composition for thetreatment of malaria comprising a bioactive water extract of Gomphostemaniveum and one or more pharmaceutically acceptable additives.

In one embodiment of the invention, the pharmaceutically acceptableadditives are selected from the group consisting of carriers diluents,stabilizing agents, solvents, flavoring agents and the like.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is the HPLC profile of the crude extract of the invention.

FIG. 2 is the total ion chromatogram of the crude extract after furtherfractionation.

DETAILED DESCRIPTION OF THE INVENTION

The increasing resistance of Plasmodium falciparum to currentlyavailable drug such as choloroquine, present a challenge in thetreatment of malaria, which still remains one of the treatment formalaria, which still remains one of the leading causes of death in theworld. Consequently new agents are urgently needed to treat theorganism.

The present invention provides a bioactive water fraction named as GN-Wobtained from the leaves of plant Gomphostemma Niveum having inhibitoryactivity against chloroquine resistant and chloroquine sensitivemalarial parasite. According to another embodiment of the presentinvention it provides the simple and efficient method of extraction,isolation and identification for the bioactive compound from the sun/airdried leaves of Gomphostemma Niveum is provided.

Further in accordance with another embodiment of the present inventionis the development of fingerprinting high performance liquidchromatographic (HPLC) method for the routine monitoring of the crudeextract. Additionally this invention provides a suitable technique forin vitro and in vivo bio-evaluation of crude extract.

Yet another embodiment of the present invention, synergistic effect hasalso been investigated in case of in vitro and in vivo anti-malarialbioassay of the crude extract. This invention also presents thesolubility and temperature related stability studies for the bioactivefraction. Evaluation of various routes of administration their stabilityin body fluid, toxicity, capacity to reach infected tissue in anadequate concentration during in vitro bioassay has also been describedby the present invention. Hence the present invention provides apharmaceutical formulation named as GN-W which exhibit excellentanti-malarial activity.

Although the invention has been described in detail with reference to apreferred embodiment it is to be understood that the above descriptionof the present invention is susceptible to considerable modifications,variations and adaptations by those skilled in the art, such intended tobe considered to be within the scope and spirit of present invention.

The detailed description of the invention which provides a bioactivewater fraction named as GN-W obtained from the plant Gomphostema niveumhaving inhibitory activity against chloroquine resistant and chloroquinesensitive malarial parasite.

The process of the bioactive water fraction is now shown with anexample, which is illustrative and is not intended to be takenrestrictively to imply any limitation on the scope of the presentinvention.

The process comprised of the following steps:

Step 1

In our efforts to isolate and identify more potent naturally occurringcompounds which are active against chloroquine resistant malarialparasite, we carry out a systematic bio-guided fractionation of thewater extract prepared from the leaves of Indian medicinal plantGomphostemma Niveum. Plant material (leaves) is collected in the monthof June and September from Dhemaji Assam located in the North Easternpart of India with the help of Defence Research Laboratory, Texpur.Leaves are dried in shade for forty eight hours and ground to finepowder.

Step 2

Extract the 100 gm of dry leaves with 500 ml of triple distilled waterusing soxhlet extraction method at reflux temperature (100° C.) foreight hours. Three repeated extractions are carried out by using 500 mlof triple distilled water respectively. The extracts are then filtered,pooled together and concentrated to dryness under reduced pressure (at86 psi and 46° C.) using the rotary evaporator. Concentrated fractionnamed as GN-W (25 gm).

Step 3

The water extract GN-W (25 gm) is further fractionated by liquid—liquidextraction using different solvents like hexane (200 ml), diethyl ether(200 ml), chloroform (200 ml) and ethanol (200 ml) on the basis of theirincreasing polarity. All the extracts are pooled together separately andconcentrated to dryness under reduced pressure (150 psi and 30° C.) andnamed as GN-E (10 gms), GN-C (2 gm) and GN-ET (6 gm). All thesefractions are tested against inhibition studies of malarial parasite.The result indicates that the ether/chloroform fraction exhibits maximumbioactivity against the malarial parasite. The percentage inhibition forether/chloroform fraction is 86-90%. An interesting feature can beobserved that the ethanolic fraction does not inhibit the growth ofmalarial parasite. Hence we hypothesize that the ether extract mightcontain some active principles which are able to inhibit the growth ofmalarial parasite. Therefore, to isolate the biologically activeprinciple column chromatography is carried out for the ether/chloroformfraction (GN-E). The list of extracts prepared and the laboratory courseare shown in table 1.

TABLE 1 Plant Name and part Solvent Code Gomphostema Niveum Leaves WaterGN-W Hexane GN-H Diethyl ether GN-E Chloroform GN-C Ethanol GN-ETStep 4

To purify the active principle from the ether/chloroform extract silicagel column chromatography is performed. Any person who is skilled in theart of related subjects can perform technique. About 10 g of etherfraction is packed on to a silica gel (100 gm), 200-400 mesh) columnfractions are subsequently analyzed for their in vitro inhibitoryactivity against the P. falciparum. Result indicates that the columnfractions elutes using 50-60% hexane in ethyl acetate is able to inhibitthe malarial parasite and therefore process the ability to cure malaria.Amount of solvents use for the elution of different components from theether fraction are as follows: Hexane 100-500 ml (1-8 fractions), 10-20%ethyl acetate in hexane 25-50 ml (1-2 lit), 40-50% ethyl acetate inhexane 25-50 ml (1-2 lit) 50-60% ethyl acetate in hexane 25-50 ml(1.5-2.5 lit).

Step 5

Parallel extraction is also carry out by taking 50-60 gm of leaves usinghexane (100-200 ml), diethyl ether/chloroform (100-200 ml), chloroform(100-200 ml) and ethyl alcohol (100-200 ml) at reflux temperature andalso at room temperature for 8-10 hours. The extracts are then filtered,pooled together and concentrated to dryness under reduced pressure (at150-200 psi and 30° C.) using the rotatory evaporator. Concentratedfractions are compared with respect to their HPLC profile and alsoevaluate in vitro.

Step 6

The separation of the constituents present in the extracts was carriedout on Waters HPLC instrument with UV detector. A finger printing methodwas also developed for the routine monitoring of the crude extract. TheHPLC conditions are as follows:

Column: NOVAPAK C-18, 150 × 3.9 MM, 5 MICRON Mobile phase:Acetonitrile:Water (60:40) Detector λ: 254 NM Pump: water's LC-600 UVDetector: Water's 486 Reversed Phase Isocratic system Flow rate: 1MI/MinStep 7Liquid Chromatography and Mass Spectrometric Analysis of Bioactive EtherFraction (GN-E).An aliquot of the sample is accurately weighed for dilution to a finalanalytical concentration of 100 μg/ml. In some cases however the entirecontent of unspecified quantity has to be reconstituted in 1 ml ofappropriate solvent as specified in the sample submission details anddiluted 100:1 for analysis.LC Conditions (Isocratic)

-   Column: Waters Symmetry C18 2.1×100 mm-   Mobile Phase: A=Water B=Acetonitrile-   Gradient: Isocratic 60/40 A:B-   Flowrate: 400 μI min split 4:1 into source-   Injection Volume: 20 μI-   U. V. Detector: Diode Array 220-300 nm (254 nm extracted)    LC Conditions (Gradient)-   Column: Waters Symmetry C18 2.1×100 mm-   Mobile Phase: A=Water+0.1% Formic Acid    -   B=Acetonitrile+0.1% Formic Acid-   Gradient: 5% to 95% B in 15 mins, hold to 20 mins then    re-equilibrate to 5% B at 30 mins-   Flow rate: 400 μI min split 4:1 into source Injection Volume: 20 μI-   U. V. Detector: Diode Array 220-300 nm (254 nm extracted)    MS Conditions-   Ion Mode: Electrospray +ve-   Acquisition: 80-800 Da at Ispectrum/sec, manual pusher at 35 μsec    Real-time centroid exact mass mode.-   Lockspray: Reference Mass (Sulfadimethoxine 311.0814 Da)-   Resolution: 6,000 FWHM Sampling Cone: 40V-   Collision Gas: Argon-   Collision Energy: MS Survey=5 eV (no fragmentation)    MSMS Conditions-   Ion Mode: Electrospray +ve-   Acquisition: 80-800 Da at Ispectrum/sec, manual pusher at 35 μsec    Real-time centroid exact mass mode.-   Lockspray: Reference Mass (Sulfadimthoxine 311.0814 Da)-   Resolution: 6,000 FWHM Sampling Cone: 40V Collision Gas:-   Collision Energy: 10 eV to 40 eV (optimized for individual    components)    Step 8    In Vitro Evaluation of Anti-malarial Activity

Two strains of chloroquine sensitive strain and one strain of P.falciparum isolate from patients from Jagadalpur region of the countryis adapted to and maintained in vitro. The cultures are maintained asper the standard culture procedures described earlier. The parasites aregrown in O +ve human RBCs with the addition of RPMI 1640 culture mediawith 10% Himan serum as supplement. The cells are incubated at 37° C. at5% CO₂ atmosphere and the parasitemia are checked after 24 hrs and mediachanged. When parasitemia exceeded 10% parasitized cells the culturedwith the addition of fresh RBC. The parasite growth synchronizes by thesorbitol lysis method and synchronized ring stage parasites are used fortesting. The in vitro testing is done in 100 μI complete media per wellwith the addition of 10 μl of erythrocytes with 2% of ring stages ofparasites. All the tests are run in duplicates with in 96 well flatbottomed tissue culture plate and double dilutions are made for each ofthe test compound with individual control wells only with the RPMI 1640and human serum supplement. The growth of the parasites in the presenceof each of the test compound, Chloroquine and control wells aremonitored by the examination of the giemsa stained blood smears madeafter 24 hrs of incubation. The counting is done for the presence ofmature schizonts among 200 asexual parasites and the average schizontmaturation inhibition is calculated by the formula (1-N_(t)/N_(c))×100where in N_(t) and N_(c) represent the number of schizont present in thetest and control respectively. The IC 50, IC 90 and IC 99 values arecalculated by using the commercial statistical package Sigmastat.

Invitro antimalarial inhibition studies Compound IC₅₀ IC₉₀ GN-W 153.23μg/ml 752.29 μg/ml GN-E 103.53 μg/ml 388.24 μg/ml Chloroquine  0.025 μM 0.046 μM

1. A therapeutically effective amount of an extract of the leaves ofGomphostemma niveum used for treating malaria.
 2. A method for preparingthe extract of claim 1, comprising air drying leaves of Gomphostemmaniveum, powdering the air dried leaves and subjecting the powder toextraction with water, filtering the extract and concentrating thefiltered extract to dryness under reduced pressure to obtain theextract.
 3. The method as claimed in claim 2, wherein the waterextraction is carried out three times.
 4. The method as claimed in claim2, wherein each extraction is carried out by a Soxhlet extraction methodat reflux temperature for eight hours.
 5. The method as claimed in claim2, wherein each extraction is carried out using triple distilled water.6. The method as claimed in claim 2, wherein the filtered extract isconcentrated to dryness under reduced pressure of about 86 psi at atemperature of about 46° C. using a rotary evaporator.
 7. The method asclaimed in claim 6, wherein the water extract is further fractionated byliquid—liquid extraction using solvents selected from the groupconsisting of hexane, diethyl ether, chloroform and ethanol on the basisof increasing polarity, the extracts then being concentrated to drynessunder reduced pressure of about 150 psi and at a temperature of about30° C.
 8. A pharmaceutical composition for the treatment of malariacomprising a therapeutically effective amount of a bioactive waterextract of Gomphostemma niveum and one or more pharmaceuticallyacceptable additives.
 9. The composition as claimed in claim 8, whereinthe pharmaceutically acceptable additives are selected from the groupconsisting of carriers, diluents, stabilizing agents, solvents,flavoring agents, binders, and lubricants or a mixture thereof.
 10. Thepharmaceutical composition as claimed in claim 8, wherein thecomposition is compounded with honey.